Synthetic peptide sequences useful in biological and pharmaceutical applications and methods of manufacture

ABSTRACT

The present invention relates to novel synthetic antigens, conjugates and antibodies based upon specific peptide sequences and the production thereof. More particularly, the invention relates to a polypeptide which is a determinant site of a protein, the polypeptide having from 8 to 20 amino acid residues, having an amino-terminal amino acid and a carboxyl-terminal amino acid, wherein the polypeptide includes: 
     (a) a four amino acid sequence which corresponds to the four amino acid sequence of a β-turn of the protein; 
     (b) a sequence of two to eight amino acid residues attached to the amino terminal (H 2  N--) of the four amino acid sequence; and 
     (c) a sequence of two to eight amino acid residues attached to the carboxyl terminal (--COOH) of the four amino acid sequence, 
     wherein the amino acid residues of subparts (b) and (c) correspond to those attached to the amino terminal and the carboxyl terminal, respectively of the β-turn of the protein, and the pharmaceutically acceptable salts of the polypeptide. The invention relates to a conjugate which comprises the polypeptide described above with a macromolecular carrier. The invention also relates to antibodies and the production thereof which are specific for the polypeptide or the conjugate of the peptide described above. 
     The synthetic polypeptide sequences, peptide conjugates and antibodies thereof are useful as antigens in the production of vaccines, antiviral agents, diagnostic reagents and the like, for the detection and treatment of infectious and immune diseases such as polio and cancer, and the like in mammals, particularly human beings.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the production of novel syntheticpeptide (polypeptide) sequences based upon information derived fromnatural peptide sequences and the use of these peptide sequences asantigens in the production of vaccines, antiviral agents, diagnosticreagents and the like; for the treatment of infectious and immunediseases, such as cancer, hoof and mouth disease and the like inmammals, specifically cattle and human beings. The process includes theprediction of the antigenic determinant site (β-turn) in a protein;chemically synthesizing the peptide sequences; optionally binding thepeptide to a high molecular weight carrier, such as a protein (BSA,thyroglobulin, KLH bovine gamma globulin and the like); introducing thepeptide into a host as the peptide or optionally its conjugate; andimmunologically producing antibodies to the peptide.

2. State of the Art

The development of synthetic peptides useful in biological applications,such as synthetic vaccines, has been under investigation for many years.The selection of and chemical or biological production of specificpeptide sequences has received particular attention.

Protein structure can be visualized as a hydrophobic core of amino acidssurrounded by a shell of more polar amino acids which are accessible tothe solvent at the surface of the molecule. In protein molecules whichinteract with a receptor, such as protein hormones, the interactionbetween the protein and the receptor must take place at thesurface-accessible sites while the hydrophobic core provides the threedimensional stability of the molecule. By arranging the critical bindingsite residues in the appropriate conformation, it is possible tosynthesize small fragments of the protein molecule which mimic theessential surface features of the present protein and retain theappropriate biological activity. The same criteria for choosing possiblebinding regions of protein molecules, the most important being surfaceexposure and appropriate conformation can also be used to predictantigen binding sites.

Chou and Fasman, for example, reported in Biochemistry, vol. 13, pp212-245 in 1974 that protein structures determined by x-raycrystallography were analyzed, and they then calculated theprobabilities that each of the normal amino acid residues would be in anβ-helix, β-pleated sheet or a β-turn type of structure. From theseprobability coefficients, they developed a method for predicting proteinstructure. For instance, the Chou-Fasman method for predicting thesecondary structure (including α-helix and β-pleated sheet residues) forfibroblast (F) and leukocyte (Le) interferons from the amino acidsequences is described by T. Hayes in Biochem. and Biophys. Res. Comm.,Vol. 95, No. 2, pp 872-9, published in 1980.

Several other methods have been applied to the prediction of thesecondary structure of proteins. For example, the methods ofBurgess/Sheraga (Israel J. Chem., Vol. 12, pp 239-286, published in1974, and V. I. Lim (J. Mol. Biol., vol. 88, pp 857-894, published in1974) have been used in a similar manner.

A computer program (SOAP) that progessively evaluates the hydrophilicityand hydrophobicity of a protein along its amino acid sequence isdescribed by J. Kyte and R. F. Doolittle, J. Mol. Biol., Vol. 157, pp105-132 (1982), which is incorporated herein by reference.

T. Hopp has reported that the most hydrophilic portion of a proteinmolecule will be the most exposed on the surface of a protein and willconstitute an antigenic determinant. Only the most hydrophilic regionwas reported to be an antigenic determinant (Immuno., Vol. 76, No. 6, pp3824-28, published June 1981). Hence, this predictive technique is saidto yield only one antigenic determinant on the molecule. In a morerecent publication, Hopp retracted this claim, see Genetic EngineeringNews, vol. 1, p 1 (1981).

Another group has reported that the same approach, i.e. calculation ofregions of minimum hydrophobicity (therefore, maximum hydrophilicity)using essentially the program of Kyte and Doolittle, predicts antigenicdeterminants. (See, for example, R. A. Lerner, et al., Cell., Vol. 23,pp 309-310, published in 1981). In practice the latter group synthesizedoverlapping fragments constituting the entire length of the proteinchain.

The preparation of antigenic hapten-carrier conjugates has beendiscussed by B. F. Erlanger in Methods in Enzymology, Vol. 70, pp.85-104, published in 1980 by Academic Press, Inc. of New York, N.Y.; andthe structure and specificity of synthetic polypeptide antigens isdiscussed by M. Sela in the Ann. N.Y. Acad. Sci., Vol. 169, pp 23-35(1970).

The problems associated with the development of synthetic polypeptidevaccines is discussed by A. J. Zuckerman in Nature, Vol. 295, pp 98-9,published Jan. 14, 1982; and by N. Wade in Science, Vol. 213, pp 623-8,published Aug. 7, 1981.

G. R. Dreesman et al., in Nature, vol. 295, pp 158-160, published onJan. 14, 1982, discusses the selection and preparation of syntheticpolypeptides which elicit an antibody response for hepatitis B surfaceantigen in mice after a single injection. The amino acid sequencesdescribed are different from the sequences of a natural protein.

Antibodies specific for the amino and carboxyl-terminal portions ofsimian virus 40 large T antigen are obtained by the immunization ofrabbits with synthetic peptides corresponsing to these regions. Theprocedures used in the preparation of antibodies specific for the endsof large T antigen with synthetic peptides as antigens are discussed byG. Walter et al., Proc. Natl. Acad. Sci. USA, Vol. 77, No. 9, pp5197-5200, published in September, 1980.

The synthesis of antigenically active polypeptides and a process fortheir manufacture are described in U.S. Pat. Nos. 4,327,075 and4,193,915, which are incorporated herein by reference. Additionalrelated information is also found in the foreign patent literature;e.g., EPO Patent Nos. 13 828 and EPO 44 710, which are incorporatedherein by reference.

This invention will provide a cheaper, more efficient method ofproducing biologically active polypeptides or their conjugates which areuseful in the diagnosis and treatment of diseases in mammals. Further,the synthetic vaccines containing polypeptides described herein would bepurer and safer than conventional vaccines, which presently consist ofthe killed or attenuated whole virus and sometimes debris from theculture medium as well. Such synthetic vaccines will also be safer tomanufacture since the risk of contamination of the site with pathogenswill not be present.

SUMMARY OF INVENTION

In one aspect this invention is concerned with a polypeptide, which is adeterminant site of a protein, the polypeptide having from 8 to 20 aminoacid residues, having an amino-terminal amino acid and acarboxyl-terminal amino acid, wherein the polypeptide includes:

(a) a four amino acid sequence which corresponds to the four amino acidsequence of a β-turn of the protein;

(b) a sequence of two to eight amino acid residues attached to the aminoterminal (H₂ N--) of the four amino acid sequence; and

(c) a sequence of two to eight amino acid residues attached to thecarboxyl terminal (--COOH) of the four amino acid sequence,

wherein the amino acid residues of subparts (b) and (c) correspond tothose attached to the amino terminal and the carboxyl terminal,respectively of the β-turn of the protein, and the pharmaceuticallyacceptable salts of the polypeptide.

In another aspect this invention concerns synthetic polypeptides havingfrom 8 to 20 amino acid residues and the pharmaceutically acceptablesalts thereof wherein:

(a) the amino acid sequence of a naturally occurring protein orpolypeptide is predicted by a prediction technique to select the fouramino acid sequence comprising a β-turn;

(b) a polypeptide containing the four amino acid sequence in a β-turnwith two to eight amino acid residues added to the amino terminal andtwo to eight amino acid residues added to the carboxyl terminal of thefour amino acid sequence containing the β-turn is synthesized;

(c) the polypeptide is optionally conjugated with a macromolecularcarrier; and

(d) the polypeptide or its conjugate substantially duplicates thesequence of a naturally occurring polypeptide specific antigenicdeterminant containing a β-turn.

In another aspect this invention is concerned with using the Chou-Fasmanpredictive technique to select the β-turn as is described herein.

In another aspect, this invention concerns the synthetic production of asynthetic polypeptide sequence, and optionally its conjugate, having aminimum of 8 and a maximum of 20 amino acids.

In another aspect, this invention concerns the polypeptide andpolypeptide conjugate and the synthetic production of a syntheticpolypeptide sequence containing a β-turn, and optionally its conjugate,having a minimum of 8, and a maximum of 20, amino acids wherein theamino-terminal amino acid of the polypeptide, H₂ N--, is blocked usingan acyl group, R--C(═O)--, particularly acetyl, CH₃ C(═O)--, Ac--.

In another aspect, this invention concerns the polypeptide andoptionally the polypeptide conjugate and the synthetic production of asynthetic polypeptide sequence containing a β-turn, and optionally itsconjugate, having a minimum of 8 and a maximum of 20 amino acids whereinthe carboxyl group --C(═O)--OH, on the carboxyl terminal amino acid, ofthe polypeptide has been converted to an amide, i.e., --C(═O)--NH₂.

In another aspect, this invention concerns the polypeptide andoptionally the polypeptide conjugate and the synthetic production of asynthetic polypeptide sequence containing a β-turn, and optionally itsconjugate, having a minimum of 8 and and a maximum of 20 amino acidswherein the amino terminal of the polypeptide is blocked using an acylgroup, R--C(═O)--, particularly acetyl, CH₃ C(═O)-- (Ac--), and thecarboxyl group on the carboxyl terminal of the polypeptide has beenconverted to an amide, i.e., --C(═O)--NH₂.

In another aspect this invention concerns a method for treatment of aninfectious or immune disease in a mammal, particularly a human being,wherein the method comprises administering to a subject in need of suchtreatment a therapeutically effective amount of the polypeptide sequenceor a pharmaceutically acceptable salt thereof or the vaccine producedtherefrom, optionally in a pharmaceutically suitable adjuvant, carrieror diluent.

In another aspect, this invention concerns a method of detecting aninfectious or immune disease in a mammal, particularly a human being,wherein the method comprises using a polypeptide, or a pharmaceuticallyacceptable salt thereof, or optionally a peptide conjugate, or apharmaceutically acceptable salt thereof as an immunogen to developantibodies in a suitable host animal. Such antibodies may also serve astherapeutic agents by "passive immunization", a technique whereinpartially purified immune sera from host animals or from hybridoma celllines may be injected into a sick animal or human and have a therapeuticeffect by binding to and neutralizing toxins or pathogenic organisms ortumor cells. Such antibodies may be used as diagnostic reagents inantibody based assays such as the ELISA or EMIT® assay orradioimmunoassay (RIA) techniques. The pure synthetic polypeptides(antigens), conjugates thereof, or antibodies prepared following thistechnique may also serve as calibration reagents in the ELISA, EMIT® orRIA techniques.

Definitions

As used herein:

"Acyl" refers to an alkyl containing carbonyl group, e.g. R--C(═O)--,wherein R is an alkyl group having from 1 to 8 carbon atoms, such asmethyl, ethyl, n-propyl, isopropyl, hexyl, octyl and the like. The acylgroup usually preferred in this invention is acetyl.

"Antibody" refers to a member of a family of glycosylated proteinscalled immunogloblins, which can specifically combine with an antigen.Hence, the term "antibody" is a functional term, and may also be theplural form "antibodies."

"Antigen" refers to a compound which will produce antibody formationwithout chemical modification.

"Antigentic determinant site" refers to the actual site of antibodyrecognition of the antigen. The term is also used interchangeably with"epitope".

"β-turn" (also referred to as β-bend or reverse turn) refers to astructural feature of peptides and proteins involving four consecutiveamino acid residues, hallmarked by the folding back on itself of thepeptide chain and the presence of an intramolecular hydrogen bond, seeJ. A. Smith and L. G. Pease, CRC Critical Reviews in Biochemistry, vol.8, pp 317-400, (October 1980), which is incorporated herein byreference.

"Carrier" usually refers to a high molecular weight (macromolecular)polymeric material (usually a protein), which alone or when conjugatedwith a polypeptide (hapten) will produce antibody formation. It may alsobe referred to as a substrate or protein substrate upon which an enzymeacts.

"Conjugate" refers to a polypeptide (or hapten) chemically-bonded to ahigh molecular weight (macromolecular) carrier or (substrate), such asthe prolactin antigen-bovine gammaglobulin conjugate formed by methodsknown in the art.

"ELISA" refers to an enzyme-linked-immunosorbent assay which employs anantibody or antigen bound to a solid phase and an enzyme-antigen orenzyme-antibody conjugate to detect and quantify the amount of antigenor antibody present in a sample. A description of the ELISA technique isfound in Chapter 22 of the 4th Edition of Basic and Clinical Immunologyby D. P. Sites et al., published by Lange Medical Publications of LosAltos, Calif. in 1982, and in U.S. Pat. Nos. 3,654,090; 3,850,752; and4,016,043, which are all incorporated herein by reference.

"EMIT®" refers to an enzyme-multiplied immunoassay technique which uses(1) an enzyme-labeled hapten, (2) specific antibody to the hapten, (3)pretreatment reagent, (4) buffered-enzyme substrate and (5) standards todetect the amount of an unknown in a sample. A description of the EMITtechnique is found in Enzyme Immunoassay, edited by E. T. Maggio,published in 1980 by CRC Press, Inc., Boca Raton, Fla., particularly onpp. 141-150, 234-5 and 242-3.

"Enzyme" refers to a protein capable of accelerating or producing bycatalytic action some change in a substrate for which it is oftenspecific. Enzymes are divided into six main groups: oxidoreductases,transferases, hydrolases, lyases, isomerases and ligases.

"Enzyme labeled" refers to an enzyme which has been further labeled witha means of detection, such as a fluorescent molecule, a radioactivegroup or the like.

"Epitope" refers to that portion of a molecule which is specificallyrecognized by an antibody. It is also referred to as the determinantsite or antigenic determinant site.

"Hapten" refers to a compound, usually of low molecular weight, forwhich antibodies can be formed by bonding of the compound to anothercompound or material, such as a protein, which has antigenic properties.

"Ligand" refers to any molecule which can bind to a receptor, for ourpurposes an antibody combining site. Such a molecule is normallyantigenic or haptenic.

"Lower alkyl" refers to a straight or branched chain saturatedhydrocarbon group having from 1 to 4 carbon atoms such as, for example,methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl andtert-butyl.

"Peptide" or "polypeptide" refers to any member of a class of compoundsof relatively low molecular weight which yield two or more amino acidson hydrolysis. Formed by the loss of water from the --NH₂ and --COOHgroups of adjacent amino acids, they are known as di-, tri-, tetra-(etc.) peptides, depending on the number of amino acids in the molecule.Peptides form the constituent parts of proteins, and may be linear orcyclic.

"Pharmaceutically acceptable salt" refers to salts that retain thedesired biological activity of the parent compound and do not impart anyundesired toxicological effects. Examples of such salts are (a) acidaddition salts formed with inorganic acids, for example hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid andthe like; and salts formed with organic acids such as, for example,acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid,fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid,benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonicacid; (b) salts with polyvalent metal cations such as zinc, calcium,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and thelike; or with an organic cation formed from N,N'-dibenzylethylenediamineor ethylenediamine; or (c) combinations, of (a) and (b), e.g. a zinctannate salt and the like.

"Protein" refers to any one of a group of complex organic nitrogenouscompounds, widely distributed in plants and animal tissue. Proteins areare essentially combinations of amino acids in peptide linkages.

"Radioimmunoassay" or "(RIA)" refers to an antibody based assay in whichthe ligand to be measured displaces or competes for binding with aradio-labelled ligand in an antibody-ligand complex. The complex isseparated and the percentage of radio-ligand bound gives a measure ofthe amount of assayable cold (non-radioactive) ligand.

"Receptor" for our purposes refers to a specific region of an antibodywith the capability of combining specifically with an antigen. It canalso refer to a protein which can bind other ligands and thereby causebiological effects.

"Vaccine" usually refers to a suspension or solution of attenuated orkilled microorganisms (bacteria, viruses, etc.) or syntheticpolypeptides or conjugates administered for the prevention, ameliorationor treatment of infectious diseases via stimulation of the production ofspecific antibodies against the disease causing organism. In thisapplication, vaccine also includes the polypeptide sequences andoptionally the polypeptide conjugates described and claimed herein foruse in both active and passive immunization.

The definitions of additional terms in this area may be found inDorland's Illustrated Medical Dictionary, 25th Ed., published by W. B.Saunders Company of Philadelphia, Pa. in 1974, and an overview ofdiagnostic applications of enzyme receptors is found in EnzymeImmunoassay, edited by E. T. Maggio, supra, both of which areincorporated herein by reference.

As set forth above and for convenience in describing this invention, theconventional abbreviations for the various common amino acids are usedas generally accepted in the peptide art as recommended by the IUPAC-IUBCommission on Biochemical Nomenclature, Biochemistry, 11, 1726 (1972)and represent L-amino acids with the exception of the achiral amino acidglycine. All peptide sequences mentioned herein are written according tothe generally accepted convention whereby the N-terminal (oramino-terminal) amino acid is on the left and the C-terminal(carboxyl-terminal) amino acid is on the right.

As used herein, the following abbreviations are used for the amino acidsdescribed in this application:

    ______________________________________                                               Alanine         Ala                                                           Arginine        Arg                                                           Asparagine      Asn                                                           Aspartic Acid   Asp                                                           Cysteine        Cys                                                           Glutamine       Gln                                                           Glutamic Acid   Glu                                                           Glycine         Gly                                                           Histidine       His                                                           Isoleucine      Ile                                                           Leucine         Leu                                                           Lysine          Lys                                                           Methionine      Met                                                           Phenylalanine   Phe                                                           Proline         Pro                                                           Serine          Ser                                                           Threonine       Thr                                                           Tryptophan      Trp                                                           Tyrosine        Tyr                                                           Valine          Val                                                    ______________________________________                                    

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Preferred embodiments of the present invention include the polypeptideshaving following the amino acid sequences, ##STR1## and thepharmaceutically acceptable salts thereof.

Subgroups of the above embodiments include the polypeptide describedwherein the amino-terminal amino acid is blocked using an acyl group.

Subgroups of the above embodiments include the polypeptide describedwherein the carboxyl-terminal amino acid has been converted to an amidegroup.

Subgroups of the above embodiments include the polypeptide describedwherein the amino-terminal amino acid is blocked using an acyl group andthe carboxyl-terminal amino acid has been converted to an amide group,respectively.

An embodiment of the present invention includes a conjugate whichcomprises a polypeptide described herein conjugated with amacromolecular carrier.

A preferred subgroup includes the conjugate wherein the macromolecularcarrier is selected from the group consisting of polysaccharides,polymeric amino acids and the copolymers thereof, enzymes and proteins,particularly an enzyme.

Preferred conjugates include those of the polypeptides specificallydescribed hereinabove.

A preferred subgroup includes the conjugate, wherein in the polypeptidethe amino-terminal amino acid is blocked using an acyl group,particularly an acetyl group.

A preferred subgroup includes the conjugate wherein in the polypeptidethe carboxyl-terminal amino acid has been converted to an amide group.

A preferred subgroup includes the conjugate wherein in the polypeptide,the amino-terminal amino acid is blocked using an acyl group and thecarboxyl-terminal amino acid has been converted to an amide group,respectively.

An embodiment of the present invention includes a method of producingthe polypeptide described herein which comprises adding from two toeight amino acid residues to each of the carboxyl-terminal amino acidand amino-terminal amino acid, respectively, of a four amino acidsequence having a β-turn of a naturally occurring protein orpolypeptide.

A subgroup includes the method wherein the four amino acid sequence ofthe β-turn is determined to have a β-turn by a prediction technique,particularly the Chou-Fasman technique.

An embodiment of the present invention further includes the methoddescribed above which includes the step of conjugating the polypeptidewith a macromolecular carrier.

An embodiment of the present invention includes a method of producing anantibody specific for the polypeptide or conjugate described above whichcomprises challenging a host animal with the polypeptide or theconjugate to elicit the production of the antibody and collecting theantibody.

An embodiment of the present invention includes an antibody specific forthe conjugate described hereinabove, and for a protein containing, as adeterminant site, a polypeptide sequence which corresponds to the β-turnsequence of the polypeptide.

Preferred subgroups include the method of producing conjugates of thepolypeptides described herein, and the pharmaceutically acceptable saltsthereof.

An embodiment of the present invention includes an antibody specific forthe polypeptide described herein, and for a protein containing, as adeterminant site, a polypeptide sequence which corresponds to the β-turnsequence of the polypeptide.

An embodiment of the present invention includes an antibody specific forthe conjugate described herein, and for a protein containing, as adeterminant site, a polypeptide sequence which corresponds to the β-turnsequence of the polypeptide.

A method for treating an infectious or immune disease in a mammal whichcomprises administering to a subject in need of such treatment atherapeutically effective amount of the polypeptide or conjugatedescribed herein or a pharmaceutically acceptable salt thereof,optionally in a pharmaceutically suitable adjuvant.

A method for treating an infectious or immune disease in a mammal whichcomprises administering to a subject in need of such treatment atherapeutically effective amount of the antibody of the polypeptide orconjugate described herein above or a pharmaceutically acceptable saltthereof, optionally in a pharmaceutically suitable adjuvant.

A method for treating an infectious or immune disease in a mammal whichcomprises administering to a subject in need of such treatment atherapeutically effective amount of the antibody described herein or apharmaceutically acceptable salt thereof, optionally in apharmaceutically suitable adjuvant.

The method of treating infectious or immune disease wherein the mammalis a human being.

An embodiment of the present invention includes a pharmaceuticalcomposition useful for treating an immune or infectious disease whichcomprises a therapeutically effective amount of the polypeptide orpolypeptide conjugate described herein above in admixture with apharmaceutically acceptable adjuvant.

A pharmaceutical composition useful for treating an immune or infectiousdisease which comprises a therapeutically effective amount of theantibody of the polypeptide or conjugate described herein in admixturewith a pharmaceutically acceptable adjuvant.

Subgroups include the specific polypeptides or polypeptides conjugatesdescribed herein.

A method for determining the presence in a sample of a protein having,as a determinant site, a polypeptide sequence which corresponds to theβ-turn sequence of the polypeptide described herein, which methodcomprises:

(a) combining the sample in an aqueous medium with the antibody of thepolypeptide or conjugate described herein and the conjugate of an enzymeas described herein;

(b) determining the enzyme activity of the combination, which activityis related to the presence of the protein in the sample.

A subgroup includes the method which further includes the comparing theenzyme activity of step (b) immediately above with the enzyme activityof an assay medium having a known amount of the protein.

A composition useful in the comparison of the enzyme activity of step(b) above, as the assay medium having a known amount of the protein,which composition comprises an assay medium having a known amount of thepolypeptide described herein.

An immunoassay method for determining the presence of a proteincontaining, as a determinant site, a polypeptide sequence whichcorresponds to the β-turn sequence of the polypeptide described herein,which method includes employing as a reagent an antibody specific forthe protein or a labeled antibody specific for the protein, theimprovement which comprises employing the antibody of the polypeptidedescribed herein as the antibody specific for the protein or as thelabeled antibody specific for the protein.

A kit for an assay for the determination of a protein having, as adeterminant site, the polypeptide sequence which corresponds to theβ-turn sequence of the polypeptide described herein in a samplecontaining such protein, which kit includes as a reagent in such assay,in a packaged combination in predetermined ratio, an antibody specificfor the protein, or a labeled antibody specific for the protein, theimprovement which comprises employing the antibody of the polypeptidedescribed herein as the antibody specific for the protein.

PROCESS FOR PREPARATION Selection of the Antigentic Determinant Site

A number of methods dealing with methods for empirical prediction ofprotein structure and their applications have been published, see, forexample, B. W. Matthews, Biochim. Biophys. Acta, vol. 405, p 442 (1975)which is incorporated herein by reference.

The Chou and Fasman (supra) correlative method is a widely usedprocedure for predicting the conformation of the peptide backbone inproteins. As part of this technique, Chou and Fasman established a setof numerical values to predict those amino acid sequences which wouldhave a probability of being in a β-turn. See Table 1 and P. Y. Chou andG. E. Fasman, Adv in Enzymology, vol. 47, pp 45-147 (1978), which isincorporated herein by reference.

                                      TABLE I                                     __________________________________________________________________________    Frequency Hierarchies of Amino Acids in the β-Turns of 29                Proteins.sup.a                                                                i     i + 1 i + 2   i + 3                                                                             P.sub.t                                                                              Pt.sub.2                                       __________________________________________________________________________    Asn                                                                              0.161                                                                            Pro                                                                              0.301                                                                            Asn                                                                              0.191                                                                            Trp                                                                              0.167                                                                            Asn                                                                              1.56                                                                              Pro                                                                              2.04                                        Cys                                                                              0.149                                                                            Ser                                                                              0.139                                                                            Gly                                                                              0.190                                                                            Gly                                                                              0.152                                                                            Gly                                                                              1.56                                                                              Gly                                                                              1.63                                        Asp                                                                              0.147                                                                            Lys                                                                              0.115                                                                            Asp                                                                              0.179                                                                            Cys                                                                              0.128                                                                            Pro                                                                              1.52                                                                              Asp                                                                              1.61                                        His                                                                              0.140                                                                            Asp                                                                              0.110                                                                            Ser                                                                              0.125                                                                            Tyr                                                                              0.125                                                                            Asp                                                                              1.46                                                                              Asn                                                                              1.56                                        Ser                                                                              0.120                                                                            Thr                                                                              0.108                                                                            Cys                                                                              0.117                                                                            Ser                                                                              0.106                                                                            Ser                                                                              1.43                                                                              Ser                                                                              1.52                                        Pro                                                                              0.102                                                                            Arg                                                                              0.106                                                                            Tyr                                                                              0.114                                                                            Gln                                                                              0.098                                                                            Cys                                                                              1.19                                                                              Lys                                                                              1.13                                        Gly                                                                              0.102                                                                            Gln                                                                              0.098                                                                            Arg                                                                              0.099                                                                            Lys                                                                              0.095                                                                            Tyr                                                                              1.14                                                                              Tyr                                                                              1.08                                        Thr                                                                              0.086                                                                            Gly                                                                              0.085                                                                            His                                                                              0.093                                                                            Asn                                                                              0.091                                                                            Lys                                                                              1.01                                                                              Arg                                                                              1.05                                        Tyr                                                                              0.082                                                                            Asn                                                                              0.083                                                                            Glu                                                                              0.077                                                                            Arg                                                                              0.085                                                                            Gln                                                                              0.98                                                                              Thr                                                                              0.98                                        Trp                                                                              0.077                                                                            Met                                                                              0.082                                                                            Lys                                                                              0.072                                                                            Asp                                                                              0.081                                                                            Thr                                                                              0.96                                                                              Cys                                                                              0.92                                        Gln                                                                              0.074                                                                            Ala                                                                              0.076                                                                            Thr                                                                              0.065                                                                            Thr                                                                              0.079                                                                            Trp                                                                              0.96                                                                              Gln                                                                              0.84                                        Arg                                                                              0.070                                                                            Tyr                                                                              0.065                                                                            Phe                                                                              0.065                                                                            Leu                                                                              0.070                                                                            Arg                                                                              0.95                                                                              Glu                                                                              0.80                                        Met                                                                              0.068                                                                            Glu                                                                              0.069                                                                            Trp                                                                              0.064                                                                            Pro                                                                              0.068                                                                            His                                                                              0.95                                                                              His                                                                              0.77                                        Val                                                                              0.062                                                                            Cys                                                                              0.053                                                                            Gln                                                                              0.037                                                                            Phe                                                                              0.065                                                                            Glu                                                                              0.74                                                                              Ala                                                                              0.64                                        Leu                                                                              0.061                                                                            Val                                                                              0.048                                                                            Leu                                                                              0.036                                                                            Glu                                                                              0.064                                                                            Ala                                                                              0.66                                                                              Phe                                                                              0.62                                        Ala                                                                              0.060                                                                            His                                                                              0.047                                                                            Ala                                                                              0.035                                                                            Ala                                                                              0.058                                                                            Met                                                                              0.060                                                                             Met                                                                              0.51                                        Phe                                                                              0.059                                                                            Phe                                                                              0.041                                                                            Pro                                                                              0.034                                                                            Ile                                                                              0.056                                                                            Phe                                                                              0.60                                                                              Trp                                                                              0.48                                        Glu                                                                              0.056                                                                            Ile                                                                              0.034                                                                            Val                                                                              0.028                                                                            Met                                                                              0.055                                                                            Leu                                                                              0.59                                                                              Val                                                                              0.43                                        Lys                                                                              0.055                                                                            Leu                                                                              0.025                                                                            Met                                                                              0.014                                                                            His                                                                              0.054                                                                            Val                                                                              0.50                                                                              Leu                                                                              0.36                                        Ile                                                                              0.043                                                                            Trp                                                                              0.013                                                                            Ile                                                                              0.013                                                                            Val                                                                              0.053                                                                            Ile                                                                              0.47                                                                              Ile                                                                              0.29                                        __________________________________________________________________________

The technique then multiplies the probabilities shown in the above tableof each amino acid in a particular quartet of amino acids to be atposition "i" in a β-turn times the probability of the amino acid atposition i+1 to be in position "i+1", etc.

Thus, for the sequence of amino acids 82 to 85 of FMDV-UP_(th), that is,His-Glu-Gly-Asp, the following calculation is made:

    ______________________________________                                        P.sub.turn =                                                                              f(amino acid at position i) ×                                           f(amino acid at position i + 1) ×                                       f(amino acid at position 1 + 2) ×                                       f(amino acid at position i + 3).                                  ______________________________________                                    

Then substituting the amino acids numbered 82 to 85,

    P.sub.turn =f(His)×f(Glu)×f(Gly)×f(Asp).

Next inserting the probabilities of these amino acids,

    ______________________________________                                        P.sub.turn =                                                                              f(0.14) × f(0.07) × f(0.19) ×                               f(0.081); thus                                                    ______________________________________                                    

thus

    P.sub.turn =1.5×10.sup.-4

Chou and Fasman indicate that for prediction of a β-turn by this methodthe calculated probability of a β-turn should be greater than or equalto 0.75×10⁻⁴. Therefore, a β-turn would be predicted for the amino acidsequence: His-Glu-Gly-Asp.

All possible quartets of adjacent amino acid residues in the protein areexamined in this manner, and those regions meeting the P_(turn) greaterthan or equal to 0.75×10⁻⁴ criterion are chosen as potential β-turns andtherefore biologically active epitopes. Regions on both sides of thepredicted β-turn (usually 2 to 8 amino acids, preferably 5) are thensynthetically added to provide additional stabilization to the β-turnvia hydrophobic interaction.

Our study of the criteria and analysis of protein structures leads us tothe conclusion that a particular type of protein conformation, theβ-turn, is a good predictor for regions of the molecule which will becrucial to both receptor binding and to antibody recognition. The β-turnstructure is a prominent surface feature typically appearing at the endof a hydrophobic (interior) domain. The β-turn region is not heavilyinvolved in internal hydrogen bonds, such as those found in α-helix andβ-sheet structures where all available hydrogen bond sites are usuallyoccupied. The single internal hydrogen bond stabilizing the β-turnregion leaves several interaction sites available for intermolecularhydrogen bonding between the β-turn backbone and receptor or antibodybinding sites.

The following peptide sequences from naturally occurring proteins wereanalyzed using this predictive technique: ##STR2##

The amino acids of the predicted β-turn in the natural protein aresynthesized as is described below. The 2 to 8 amino acids added to theamino terminal (H₂ N--) and the carboxyl terminal [--C(═O)--OH] of theβ-turn are also the same as is found in the natural protein sequence. Insome cases, however depending upon the desired result, one or moredifferent amino acids may be substituted directly for the natural ones,within the 2 to 8 amino acids in the non-β-turn portion of thepolypeptide.

Chemical Synthesis of the Amino Acid Sequence

The polypeptides of the present invention may be synthesized by anytechniques that are known to those skilled in the peptide art. Anexcellent summary of the many techniques so available may be found in J.M. Stewart and J. D. Young, "Solid Phase Peptide Synthesis", W. H.Freeman Co., San Francisco, 1969, and J. Meienhofer, "Hormonal Proteinsand Peptides", vol. 2, p 46., Academic Press (New York), 1973 for solidphase peptide synthesis and E. Schroder and K. Lubke, "The Peptides",Vol. 1, Academic Press (New York), 1965 for classical solutionsynthesis.

In general, these methods comprise the sequential addition of one ormore amino acids or suitably protected amino acids to a growing peptidechain. Normally, either the amino or carboxyl group of the first aminoacid is protected by a suitable protecting group. The protected orderivatized amino acid can then be either attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected, under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is then added, andso forth. After all the desired amino acids have been linked in theproper sequence, any remaining protecting groups (and any solid support)are removed sequentially or concurrently, to afford the finalpolypeptide. By simple modification of this general procedure, it ispossible to add more than one amino acid at a time to a growing chain,for example, by coupling (under conditions which do not racemize chiralcenters) a protected tripeptide with a properly protected dipeptide toform, after deprotection, a pentapeptide.

A particularly preferred method of preparing compounds of the presentinvention involves solid phase peptide synthesis.

In this particularly preferred method the α-amino function of the aminoacids is protected by an acid or base sensitive group. Such protectinggroups should have the properties of being stable to the conditions ofpeptide linkage formation, while being readily removable withoutdestruction of the growing peptide chain or racemization of any of thechiral centers contained therein. Suitable protecting groups aret-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z),biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,alpha, alpha-dimethyl-3,5-dimethoxybenzyloxycarbonyl,o-nitrophenylsulfenyl, 2-cyano-1,1-dimethyl-ethoxycarbonyl,9-fluorenylmethoxycarbonyl and the like, especially t-butyloxycarbonyl(Boc).

Particularly preferred side chain protecting groups are, for arginine:nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Z, Boc andadamantyloxycarbonyl; for tyrosine: benzyl, o-bromobenzyloxycarbonyl,2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl and acetyl; forserine: benzyl and tetrahydropyranyl; for histidine: benzyl,p-toluenesulfonyl and 2,4-dinitrophenyl.

The carboxyl-terminal amino acid (C-terminal) [--C(═O)--OH] is attachedto a suitable solid support. Suitable solid supports useful for theabove synthesis are those materials which are inert to the reagents andreaction conditions of the stepwise condensation-deprotection reactions,as well as being insoluble in the media used. Suitable solid supportsare chloromethylpolystyrene-divinylbenzene polymer,hydroxy-methyl-polystyrene-divinylbenzene polymer, and the like,especially chloromethyl-polystyrene-1% divinylbenzene polymer. For thespecial case where the carboxyl-terminal amino acid of the peptidebecomes an amide [--C(═O)--NH₂ ], a particularly useful support is thebenzhydrylamino-polystyrene-divinylbenzene polymer described by P.Rivaille, et al., Helv. Chim. Acta., 54, 2772 (1971). The attachment tothe chloro-methyl polystyrene-divinylbenzene type of resin is made bymeans of the reaction of the N.sup.α -protected amino acid, especiallythe Boc-amino acid, as its cesium, tetramethylammonium,triethylammonium, 4,5-diazabicyclo[5.4.0]undec-5-ene, or similar salt inethanol, acetonitrile, N,N-dimethylformamide (DMF), and the like,especially the cesium salt in DMF, with the chloromethyl resin at anelevated temperature, for example between about 40° and 60° C.,preferably about 50° C., for from about 12 to 48 hours, preferably about24 hours. The N.sup.α -Boc-amino acid is attached to the benzhydrylamineresin by means of an N,N'-dicyclohexylcarbodiimide(DCC)/1-hydroxybenzotriazole (HBT) mediated coupling for from about 2 toabout 24 hours, preferably about 12 hours at a temperature of betweenabout 10° and 50° C., preferably 25° C. in a solvent such asdichloromethane or DMF, preferably dichloromethane. The coupling ofsuccessive protected amino acids can be carried out in an automaticpolypeptide synthesizer as is well known in the art. The removal of theN.sup.α -protecting groups may be performed in the presence of, forexample, a solution of trifluoroacetic acid in methylene chloride,hydrogen chloride in dioxane, hydrogen chloride in acetic acid, or otherstrong acid solution, preferably 50% trifluoroacetic acid indichloromethane at about ambient temperature. Base labile protectinggroups maybe removed by treatment with a base such as piperidine in DMFor dioxane. Each protected amino acid is preferably introduced inapproximately 2.5 molar excess and the coupling may be carried out indichloromethane, dichloromethane/DMF mixtures, DMF and the like,especially in methylene chloride at about ambient temperature. Thecoupling agent is normally DCC in dichloromethane but may beN,N'-di-iso-propylcarbodimide or other carbodiimide either alone or inthe presence of HBT, N-hydroxysuccinimide, other N-hydroxyimides oroximes. Alternately, protected amino acid active esters (e.g.p-nitrophenyl, pentafluorophenyl and the like) or symmetrical anhydridesmay be used.

At the end of the solid phase synthesis, the polypeptide is eithercarried through another deprotection and neutralization cycle followedby acylation, preferably acetylation with acetic anhydride to yield anN-acetyl (N--Ac) blocked amino-terminal or it may be removed from theresin directly. If the carboxyl [--C(═O)--OH] terminal is to be blockedas the amide, the peptide may be either be synthesized on thebenzhydrylamino-polystyrene resin, which gives the amide directly or itmay be removed from the resin by ammonolysis with, for example,ammonia/methanol or ammonia/ethanol, at a temperature of from about 0°to about 50° C., preferably about 25° C. for about 12 to about 48 hr,preferably about 18 hr. If a peptide with a free amino-terminal and acarboxyl-terminal is desired, the peptide may be directly removed fromthe resin by treatment with anhydrous liquid hydrogen fluoride in thepresence of a radical scavanger such as anisole. The amino- orcarboxyl-blocked (protected) peptides, either on the resin or removedfrom the resin by ammonolysis, are similarly deprotected by treatmentwith anhydrous liquid hydrogen fluoride. In cases where base labileprotection of the N.sup.α function is used in conjunction with t-butylbased side chain protection, the final resin removal and deprotectionstep may be performed with trifluoroacetic acid.

Other means of removal of the (side chain) protecting groups from thepolypeptide are treatment with hydrogen fluoride/pyridine complex,treatment with tris(trifluoroacetyl)boron and trifluoroacetic acid, byreduction with hydrogen and palladium on carbon or polyvinylpyrrolidone,or by reduction with sodium in liquid ammonia, preferably with liquidhydrogen fluoride, and anisole at a temperature between about -10° and+10° C., preferably about 0° C., for between about 15 minutes and 1hour, preferably about 30 minutes. The latter treatment (HF/anisole) maybe used for simultaneous cleavage from the resin and deprotection toyield free --CO₂ H when a normal benzylester linkage has been used or toform a CO--NH₂ when a benzhydrylamino linkage has been used. For theamide terminal peptides on the benzhydrylamine resins, the resincleavage and deprotection steps may be combined in a single steputilizing liquid hydrogen fluoride and anisole as described above. Thefully deprotected polypeptide is then purified by a sequence ofchromatographic steps employing any or all of the following types: ionexchange on a weakly basic resin in the acetate form; hydrophobicadsorption chromatography on underivatized polystyrene-divinylbenzene(for example Amberlite XAD); silica gel adsorption chromatography; ionexchange chromatography on carboxymethylcellulose; partitionchromatography, e.g. on Sephadex G-25, or countercurrent distribution;high performance liquid chromatography (HPLC), especially reverse phaseHPLC on octyl- or octadecylsilyl-silica bonded phase column packing.

Thus in an additional aspect, the invention is concerned with free andprotected (or blocked) synthetic polypeptides having 8 to 20 amino acidresidues, i.e.; for the human growth hormone fragment:

H-[Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp]-OH (unprotected or freepolypeptide);

Ac-[Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp]-OH [amino-terminal (--NH₂)protected as Ac--NH--)];

H-[Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-]NH₂ ; [carboxyl-terminal[--C(═O)--OH] protected as the amide, --C--(═O)--NH₂ ]; and

Ac-[Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp]-NH₂ ; [amino-terminal protected asAc--NH--, and carboxyl-terminal protected as --C(═O)--NH₂ ].

(For conjugation with a high molecular weight carrier of the polypeptidewhere both the amino-terminal and the carboxyl-terminal have beenprotected, a chemically reactive group should be present within thepolypeptide chain, e.g., --OH of tyrosine, for the chemical attachmentof the conjugate.)

Thus, in another aspect the present invention relates to a method forpreparing these compounds of the which process comprises:

(i) coupling fragments; or

(ii) removing protecting groups and, optionally, covalently bound solidsupport from a protected polypeptide to afford a compound of theaforementioned polypeptide sequences or a salt thereof, and optionally

(iii) converting a compound of the aforementioned polypeptide sequencesto a pharmaceutically acceptable salt thereof, or

(iv) converting a salt of a compound of the aforementioned polypeptidesequences to a pharmaceutically acceptable different salt thereof, or

(iv) decomposing a salt of a compound of the aforementioned polypeptidesequences to a free polypeptide of the aforementioned polypeptidesequences.

CONJUGATION OF POLYPEPTIDES TO PROTEIN CARRIERS

A. Functional Groups--The polypeptides described herein may be coupledto protein or high molecular weight (macromolecular) polymeric carriers(or substrates) through several types of functional groups on theantigen, e.g. (a) α or ε-amino groups, (b) α, β, or γ-carboxyl groups,(c) thiol (--SH) groups, (d) aromatic rings (e.g., Tyr, His)

B. Specific Techniques -

1. α- or β-amino groups

Several coupling methods are available.

a. The carboxyl functional groups on a carrier may be activated withcarbodiimides (especially water soluble carbodiimides (WSC) such asN-ethyl-N'-(3-dimethylaminopropylcarbodiimide), isoxazolium salts (e.g.,Woodwards Reagent K), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline(EEDQ), active ester forming reagents (to yield N-hydroxysuccinimideesters, 1-hydroxybenzotriazole esters, nitrophenyl esters,pentafluorophenyl esters, etc.), reagents yielding acid chorides (e.g.,PCl₅, but only for non-protein carriers), reagents yielding mixedanhydrides (e.g., isobutylchloroformate, acetic anhydride, pivalicanhydride).

The free amino function of the synthetic antigen (either α-aminofunction or ε-amino function of lysine) is then allowed to react withthe activated carboxyl function of the carrier in an aqueous buffer (pHmay be from about 6.5 to 9, optimally about 8) or in a mixedorganic/aqueous buffer system (e.g., DMF/water, pH 8). For non-proteincarriers, an organic solvent (e.g., DMF) may be used. Especially usefultechniques in this class are concurrent activation of the proteincarrier with WSC and coupling with antigen in aqueous buffer orpreparation of the p-NO₂ -phenyl ester of a succinylated protein carrierfollowed by coupling with the antigen in aqueous buffer.

b. The amino function(s) on the synthetic antigen may be crosslinkedwith amino functions on the carrier molecule by reaction withgluteraldehyde in aqueous solution on mixed organic/aqueous solution(pH˜7) at room temperature.

c. The amino function(s) on the synthetic antigen may be crosslinkedwith amino functions on the carrier molecule by reaction withbifunctional crosslinking reagents such as dimethylsuberimidate,phenyldiisocyanate, phenyldiisothiocyanate, difluorodinitrobenzene, orcyanic chloride.

2. α, β, or γ Carboxyl groups

The carboxyl functions on the synthetic antigen will be activated by thetechniques listed under Section B.(1) a for caarboxyl activation. Theactivated carboxyl functions will then be reacted with the aminofunctions on a suitable carrier molecule using the aqueous or mixedorganic/aqueous buffer conditions described above.

3. Thiol (--SH) Groups

The --SH groups on the synthetic antigen (incorporated as cysteinyl orhomocysteinyl residues on or in the polypeptide chain) will be reactedwith suitable carriers which have been modified by the incorporation ofmaleimide functions. The --SH function inserts specifically into thedouble bond of the maleimide function and yields an antigen-carriercomplex in which the carrier has retained a monomeric nature. The SHfunction may be incorporated into the antigen by either incorporation ofa Cys (or homo-Cys) residue in α-amino acid linkage, or by reaction ofan amino function (α-amino or ε-amino of lysine) with cysteinethiolactone. Alternatively, the --SH function on the antigen may beactivated as the 2- or 4-thiopyridyldisulfide and bonded to the --SHgroups on a suitable carrier. This sequence may also be reversed withthe carrier --SH activated as the thiopyridyldisulfide.

4. Aromatic rings

The aromatic rings of Tyr and His may be crosslinked to the aromaticrings of Tyr and His residues of proteins by means of bis-diazotizedaromatic compounds (e.g., bis-diazotized-benzidine or bis-diazotizedo-anisidide). This reaction is performed on an aqueous or mixedorganic/aqueous solution of the antigen and carrier.

C. Suitable Protein Substrates (Carriers)

Suitable substrates (carriers) for this process, include but are notlimited to the following, large, slowly metabolized macromolecules suchas proteins; polysaccharides, such as latex functionalized sepharose,agarose, cellulose, cellulose beads and the like; polymeric amino acids,such as polyglutamic acid, polylysine and the like; and amino acidco-polymers. Especially useful protein substrates are serum albumins,tetanus toxoid, keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art. Such proteins may be used in their native form or theirfunction group content may be modified by succinylation of Lys residuesor reaction with Cys-thiolactone. A sulfhydryl group may also beincorporated into the carrier (or antigen) by reaction of aminofunctions with 2-iminothiolane or the N-hydroxysuccinimide ester of3-(4-dithiopyridyl) propionate. Suitable carriers may also have beenmodified to incorporate spacer arms (such as hexamethylene diamine orother bifunctional molecules of similar size) for attachment of theantigen.

For a recent review of these techniques see B. F. Erlanger in Methods ofEnzymology, vol. 70, p 85ff (1980), "The Preparation of AntigenicHaptene--Carrier Conjugates--A Survey".

Vaccine Preparation Techniques

Vaccine preparation techniques are generally known in the art asdescribed by J. I. Duffy (ed.) in Vaccine Preparation Techniques,published by Noyes Data Corporation of Park Ridge, N.J. in 1980, andreferences cited therein, all of which are incorporated herein byreference. More specifically, in this application the therapeutic agentof the vaccine is generally considered to be the polypeptide sequence,the polypeptide conjugate or the antibody to the polypeptide or thepolypeptide conjugate as described herein.

Introducing the Peptide Sequence into a Host and Producing Antibodies

The polypeptide sequence produced above is then introduced into amammalian host. This is usually accomplished by subcutaneous injectioninto mammals as a solution in saline which has been emulsified withcomplete Freund's adjuvant. Monthly booster injection of antigen inincomplete Freund's adjuvant are made, and the animals are bled monthly(one week prior to booster) to obtain sera for characterization.Alternatively Freud's adjuvent can be replaced by synthetic adjuvantssuch as N-Ac-muramyl-L-Ala-D-IGln or its analogs. Antibodies may beprepared by a number of known methods, see, for instance, U.S. Pat. Nos.4,082,735 and 4,082,736, which are incorporated herein by reference.)Suitable hosts include for example, monkeys, cattle, sheep, goats,swine, dogs, cats, rabbits, chickens, rats, guinea pigs, mice and thelike.

Collecting the Antibodies

The antibodies are collected by bleeding the animal, either partially(about 10% of blood volume) or by a complete exsanguination. The wholeblood is allowed to clot at 25° C. for several hrs. This aqueousammonium sulfate solution is added to achieve 40% by weight of aqueoussolution, and the IgG fraction precipitates. The clear serum isseparated from the clot by centrifugation and is diluted with 0.9%saline to the desired concentration. Alternately, the antibodies may befurther purified by affinity chromatography or salt precipitation priorto dilution. Thus, aqueous ammonium sulfate solution is added to achieve40% by weight of aqueous solution, and the IgG fraction precipitates.The precipitate is collected by centrifugation and resuspended in salineor buffer solution to the desired concentration.

The purified antibody fraction may be further modified for use indiagnostic assay systems. Such modification may encompass linkage withenzymes such as lipozyme, lactoperoxidase, et al. for use in ELISAassays. The antibody may be modified with fluorescent moieties whosefluorescence may be quenched or enhanced upon binding of the antibodyand the antigen. These techniques for assaying the extent of theantibody-antigen interaction are known in the art. The essential firststep is, however, the preparation of a suitably immunogenic polypeptide(vaccine) for administration to the animals so that a population of highaffinity antibodies will be obtained.

ANTISERUM CHARACTERIZATION

The antisera obtained are characterized by means of an ELISA assay.(see, Maggio, supra, for more detail, and U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043, which are incorporated herein by reference).Briefly, the polypeptide (antigen) or a conjugate of the polypeptide(antigen) to a non-cross reacting carrier protein, e.g., bovine serumalbumin is prepared as described above and a solution of this conjugateis dried down in individual wells of a polystyrene microwell plate. Intothese wells are placed doubling dilutions of the serum sample containingantibodies against the antigen. After an incubation period of about 8hr, the well contents are decanted, the wells are washed and acommercially available enzyme-linked goat-anti-rabbit-IgG antibodyreagent (to assay antibodies raised in rabbits) is introduced. In thecase described here the enzyme used is horseradish peroxidase. After an8 hr incubation period, the well contents are decanted and the wells arewashed. A solution containing the chromogen,2,2'-azino-di(3-ethyl-benzthiazolinesulfonic acid) ammonium salts (ABTS)and H₂ O₂, is added to the wells and the color is developed andquantitated by absorption at 415 nm in a UV spectrophotometer.Increasing optical density (OD) readings indicate greater antibodyresponse. An OD of greater than 1 indicates a moderate to strongresponse.

For example, in the case cited herein the response mounted against theprolactin fragment, H-Gly-Cys-Arg-Ile-Ile-His-Asn-Asn-Asn-Cys-OH wasmoderate with OD values measured at 3 months post-injection of 1.4 inone animal and 0.4 in another.

Chemically Synthesizing the Polypeptide in Therapeutic Quantities

After being chosen by the method described (supra) the polypeptides aresynthesized by solid phase or solution phase techniques well-known tothose skilled in the art. References to texts describing thesetechniques and general descriptions of these techniques have beenprovided (supra). Using these techniques, the desired polypeptides maybe prepared on gram, kilogram, or larger scale. More detaileddescriptions are found in the examples.

E. UTILITY AND ADMINISTRATION

In the practice of the method of this invention an effective amount of acompound of the invention or a pharmaceutical composition containingsame is administered to the subject in need of, or desiring, suchtreatment. These compounds or compositions may be administered by any ofa variety of routes depending upon the specific end use, includingparticularly parenterally (including subcutaneous, intramuscular andintravenous administration). The most suitable route in any given casewill depend upon the use, particular active ingredient, the subjectinvolved, and the judgment of the medical practitioner. The compound orcomposition may also be administered by means of slow-release, depot orimplant formulations as is well known in the art. The polypeptides,polypeptide conjugates and antibodies described herein are usuallyadministered in amounts of 1 to 100 μg per kg of body weight,particularly in amounts of 40-60 μg.

For active immunity, the polypeptide or the polypeptide conjugate isadministered to produce the antibodies in the subject in need oftreatment. In passive immunity, partially purified sera containingantibodies from host animals is introduced into the subject to produce atherapeutic effect by binding to and neutralizing the toxins, pathogenicorganisms or tumor cells.

The polypeptides, polypeptide conjugates and antibodies produced to thepolypeptides and polypeptide conjugates have a variety of uses in animaland human health disease diagnosis and treatment. These include, forexample, treatment of any disease of the immune system for whichantibodies are produced and certain cancers, such as leukemia.Infectious diseases such as hepatitis, polio, diphtheria, tetanus,typhoid and the like in humans, and calf scours and foot and mouthdisease in animals can be treated with vaccines comprised of thepolypeptides or polypeptide conjugates described herein. The vaccinesare administered in a pharmaceutically acceptable adjuvant, carrier ordiluent such as vegetable oil, as an alum precipitate in saline insynthetic oils, or even in mineral oil/with emulsion glycopeptides,and/or bacterial cell wall/fragments, and the like.

The polypeptides, polypeptide conjugates and antibodies to thesepolypeptides and polypeptide conjugates of this invention are alsouseful in the detection and diagnosis of disease, particularly inproviding high purity materials useful for calibration solutions forassay techniques, such as ELISA or EMIT.

The enzymes for use in diagnostic reagents, standards or kits can varywidely, depending on the ease of conjugation, turnover rate, and thephysiological fluid in which the unknown (analyte) is to be measured.Representative enzymes of choice include hydrolases, nucleases,amidases, esterases and the like which are found in U.S. Pat. No.3,817,837, which is incorporated herein by reference.

The methods and apparatus for labeling an antibody as described hereinfor use in a diagnostic reagent, standard or kit is found in U.S. Pat.No. 4,366,241, which is incorporated herein by reference.

The following examples serve to illustrate the invention. They shouldnot be construed as narrowing it, or limiting its scope.

EXAMPLE 1 Preparation of Human Growth Hormone FragmentH-Tyr-Gly-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-Leu-Cys-OH

In the reaction vessel of a Beckman 990 Peptide Synthesizer is placed1.06 g (1.0 mmol) of Boc-Cys(BzlOMe)-O-Resin prepared fromchloromethylpolystyrene-1%-divinylbenzene resin (1 mmol Cl/g resin) byreaction with Boc-Cys(BzlOMe)-OCs salt [(B. F. Gisin, Helv. Chim. Acta,56, 1476 (1973)]. Amino acids are added sequentially to this resin bymeans of a synthesis program, as follows:

    ______________________________________                                        Step   Action                  Time                                           ______________________________________                                        1      CH.sub.2 Cl.sub.2 wash                                                                          1     time  1.5 min                                  2      50% CF.sub.3 CO.sub.2 H/CH.sub.2 Cl.sub.2                                                       1     time  1.5 min                                         deprotection                                                           3      50% CF.sub.3 CO.sub.2 H/CH.sub.2 Cl.sub.2                                                       1     time  30 min                                          deprotection                                                           4      CH.sub.2 Cl.sub.2 wash                                                                          3     times 1.5 min                                  5      10% triethylamine/CH.sub.2 Cl.sub.2                                                             2     times 1.5 min                                  6      CH.sub.2 Cl.sub.2 wash                                                                          3     times 1.5 min                                  7      N.sup.α --Boc-amino acid                                                                  1     time  add                                             solution                                                               8      N,N'--dicyclohexylcarbo-                                                                        1     time  add                                             diimide solution                                                       9      CH.sub.2 Cl.sub.2 rinse and hold-                                                               1     time  2 hr                                            reaction coupling                                                      10     CH.sub.2 Cl.sub.2 - rinse add                                                                   1     time  1.5 min                                  11     CH.sub.2 Cl.sub.2 wash                                                                          3     times 1.5 min                                  12     ethanol wash      3     times 1.5 min                                  13     CH.sub.2 Cl.sub.2 wash                                                                          3     times 1.5 min                                  ______________________________________                                    

Steps 1-13 complete a coupling cycle for one amino acid and completenessof the reaction is checked by the ninhydrin method of E. Kaiser, et al.,Anal. Biochem., 34, 595 (1970).

The resin is coupled sequentially with about a 2.5 molar excess of eachprotected amino acid and DCC. Thus, the resin is treated duringsuccessive coupling cycles with

0.623 g of Boc-Leu-OH.H₂ O;

0.806 g of Boc-Asp(Bzl)-OH;

0.703 g of Boc-Tyr(Cl₂ Bzl)-OH;

0.543 g of Boc-Val-OH;

0.586 g of Boc-Asn-OH;

0.738 g of Boc-Ser(Bzl)-OH;

0.806 g of Boc-Asp(OBzl)-OH;

0.738 g of Boc-Ser(Bzl)OH;

0.473 g of Boc-Ala-OH;

0.438 g of Boc-Gly-OH; and

0.703 g of Boc-Tyr(Cl₂ Bzl)-OH.

The resin is removed from the reaction vessel, washed with CH₂ Cl₂, anddried in vacuo to yield 3.35 g of the protected polypeptide resin.

The polypeptide product is simultaneously removed from the resin andcompletely deprotected by treatment with anhydrous liquid HF. A mixtureof 3.34 g of protected polypeptide resin and 3.5 ml of anisole(scavenger) in a Kel-F reaction vessel is treated with 20 ml ofredistilled (from CoF₃) anhydrous liquid HF at 0° C. for 1 hour. The HFis evaporated under vacuum and the residue ofH-Tyr-Gly-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-Leu-Cys-OH, as its HF salt, iswashed with ether. The residue is then extracted with glacial aceticacid. The acetic acid extract is lyophilized to yield crude material.

Purification is achieved by preparative high performance liquidchromatography on a 205 mg sample using a 2.5×100 cm column of 20-40micron octadecylsilylated silica (Merck Lichroprep C₁₈). The eluent is83% 0.03M NH₄ OAc/17% acetonitrile. In 2 runs a total of about 400 mg ofcrude material is purified. After three lyophilizations from water, 85mg of pure H-Tyr-Gly-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-Leu-Cys-OH isobtained as the acetate salt [α]_(D) ²⁵ -20.1° (c 0.6, HOAc).

EXAMPLE 2 Preparation ofH-Tyr-Asp-Thr-Ser-Asp-Ser-Asp-Asp-Tyr-His-Leu-Cys-OH

Repeating the procedure of Example 1, using the Boc-Cys(BzlOMe)-O-Resinand substituting the following protected and unprotected amino acids:

0.623 g of Boc-Leu-OH.H₂ O;

1.023 g of Boc-His(Tos)OH;

0.703 g of Boc-Tyr(Cl₂ Bzl)OH;

0.806 g of Boc-Asp(OBzl)-OH;

0.806 g of Boc-Asp(OBzl)-OH;

0.738 g of Boc-Ser(Bzl)-OH;

0.806 g of Boc-Asp(OBzl)-OH;

0.738 g of Boc-Ser(Bzl)-OH;

0.773 g of Boc-Thr(Bzl)-OH;

0.806 g of Boc-Asp(OBzl)-OH; and

0.703 g of Boc-Tyr(Cl₂ Bzl)-OH

in the solid phase synthesis sequence, the polypeptideH-Tyr-Asp-Thr-Ser-Asp-Ser-Asp-Asp-Tyr-His-Leu-Cys-OH, is obtained ingood yield as the acetate salt [α]_(D) ²⁵ -33.4° (c 0.8, HOAc).

EXAMPLE 3 ##STR3##

Repeating the procedure of Example 1 and substituting Boc-Gly-O-Resinfor Boc-Cys(BzlOMe)-O-Resin and the following protected and unprotectedamino acids:

1.708 g of Boc-Cys(Bzl-OMe)-OH;

2.142 g of Boc-Arg-OH:

0.946 g of Boc-Ala-OH;

0.946 g of Boc-Ala-OH;

0.876 g of Boc-Gly-OH;

0.876 g of Boc-Gly-OH;

1.706 g of Boc-Pro-OH;

1.708 g of Boc-Cys(BzlOMe);

1.146 g of Boc-Ile-OH.1/2H₂ O;

1.706 g of Boc-Pro-OH; and

1.246 g of Boc-Leu-OH.H₂ O;

in the solid phase synthesis sequence, the following polypeptide:

H-Leu-Pro-Ile-Cys-Pro-Gly-Gly-Ala-Ala-Arg-Cys-Gly-OH is obtained in goodyield as the acetate salt.

A solution of 0.67 g of the reduced forms of the polypeptide in amixture of 330 ml H₂ O and 280 ml acetone at pH 6.5 is treated with 172mg of diiodoethane in 40 ml of acetone. The oxidative cyclization iscomplete after 2.5 hr. The mixture is concentrated to dryness, theresidue is dissolved in H₂ O, and the polypeptide is converted to theacetate salt by passage through an AG3 (--OAc form) weakly basic ionexchange resin (BioRad Labs, Inc.). The eluent is lyophilized to yield0.64 g of the crude disulfide. Pure product is obtained by fourprep-HPLC run on about 150-mg batches of crude material on a 2.5×100 cmcolumn of Licroprep C-18 (25-40 micron) packing material using 16% CH₃CN/84% H₂ O (0.03M in NH₄ OAc, pH 4.5) as eluent. Lyophilization yieldedthe cyclic product disulfide: ##STR4## as 66 mg of white powder, [α]_(D)²⁵ -96.8° (C 0.64, HOAc).

EXAMPLE 4 ##STR5##

Repeating the procedure of Example 3 using Boc-Cys(BzlOMe)-O-resin andthe following protected and unprotected amino acids:

1.16 g of Boc-Asn-OH;

1.16 g of Boc-Asn-OH;

1.16 g of Boc-Asn-OH;

2.052 g of Boc-His(Ts)-OH;

1.15 g of Boc-Ile-OH.1/2H₂ O;

1.15 g of Boc-Ile-OH.1/2H₂ O;

1.60 g of Boc-Arg(Ts)-OH;

1.708 g of Boc-Cys(Bzl-OMe)-OH; and

0.876 g of Boc-Gly-OH;

in the solid phase synthesis sequence, the following polypeptide:##STR6## is obtained in good yield, [α]_(D) ²⁵ -67.1° (C 0.3, HOAc).

EXAMPLE 5 Preparation of a Human Growth Hormone FragmentH-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-OH

Repeating the procedure of Example 1 and substituting aBoc-Asp(OBzl)-O-Resin for Boc-Cys(BzlOMe)-O-Resin and the followingprotected and unprotected amino acids:

0.703 g of Boc-Tyr(Cl₂ Bzl)-OH;

0.543 g of Boc-Val-OH;

0.586 g of Boc-Asn-OH;

0.738 g of Boc-Ser(Bzl)-OH;

0.806 g of Boc-Asp(OBzl)-OH;

0.738 g of Boc-Ser(Bzl)-OH; and

0.473 g of Boc-Ala-OH;

the following peptide is obtained:

H-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-OH in good yield as the acetate salt.

EXAMPLE 6 Preparation of a Human Growth Hormone FragmentH-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-Leu-Leu-Lys-Asp-Leu-Glu-OH

Repeating the procedure of Example 1 and substituting aBoc-Glu-(OBzl)-O-Resin and the following protected and unprotected aminoacids:

0.623 g of Boc-Leu-OH.H₂ O;

0.806 g of Boc-Asp-(OBzl)OH;

0.951 g of Boc-Lys(Z)-OH;

0.623 g of Boc-Leu-OH.H₂ O;

0.623 g of Boc-Leu-OH.H₂ O;

0.806 g of Boc-Asp(Bzl)-OH;

0.703 g of Boc-Tyr(Cl₂ Bzl)-OH;

0.543 g of Boc-Val-OH;

0.581 g of Boc-Asn-OH;

0.738 g of Boc-Ser(Bzl)-OH;

0.806 g of Boc-Asp-(OBzl)-OH;

0.738 g of Boc-Ser(Bzl)-OH; and

0.473 g of Boc-Ala-OH;

the following polypeptide is obtained:

H-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-Leu-Leu-Lys-Asp-Leu-Glu-OH in goodyield is the acetate salt.

EXAMPLE 7 Preparation of a Human Growth Hormone FragmentH-Asn-Ser-Leu-Val-Try-Gly-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-Leu-Leu-Lys-Asp-Leu-Glu-OH

Repeating the procedure of Example 1 and substituting a Boc-Glu-O-Resinfor Boc-Cys(BzlOMe)-O-Resin and the following protected and unprotectedamino acids:

0.623 g of Boc-Leu-OH.H₂ O;

0.806 g of Boc-Asp-(OBzl)-OH;

0.951 g of Boc-Lys-(Z)-OH;

0.623 g of Boc-Leu-OH H₂ O;

0.623 g of Boc-Leu-OH H₂ O;

0.806 g of Boc-Asp(Bzl)-OH;

0.703 g of Boc-Tyr(Cl₂ Bzl)-OH;

0.543 g of Boc-Val-OH;

0.581 g of Boc-Asn-OH;

0.738 g of Boc-Ser(Bzl)-OH;

0.806 g of Boc-Asp-(OBzl)-OH;

0.738 g of Boc-Ser-(Bzl)-OH:

0.473 g of Boc-Ala-OH;

0.438 g of Boc-Gly-OH;

0.703 g of Boc-Tyr(CH₂ Bzl)-OH;

0.543 g of Boc-Val-OH;

0.623 g of Boc-Leu-OH.H₂ O;

0.738 g of Boc-Ser(Bzl)-OH; and

0.586 g of Boc-Asn-OH;

the following polypeptide is obtained:

H-Asn-Ser-Leu-Val-Tyr-Gly-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-Leu-Leu-Lys-Asp-Leu-Glu-OHin good yield as the acetate salt.

EXAMPLE 8 Preparation of a Human Growth Hormone FragmentH-Asn-Ser-Leu-Val-Tyr-Gly-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-OH

Repeating the procedure of Example 1 and substituting aBoc-Asp-(OBzl)-O-Resin for Boc-Cys(BzlOMe)-O-Resin and the followingprotected and unprotected amino acids:

0.703 g of Boc-Tyr(Cl₂ Bzl)-OH;

0.543 g of Boc-Val-OH;

1.16 g of Boc-Asn-OH;

0.738 g of Boc-Ser(Bzl)-OH;

0.806 g of Boc-Asp-(OBzl)-OH;

0.738 g of Boc-Ser(Bzl)-OH;

0.473 g of Boc-Ala-OH;

0.438 g of Boc-Gly-OH;

0.703 g of Boc-Tyr(CH₂ Bzl)-OH;

0.543 g of Boc-Val-OH;

0.623 g of Boc-Leu-OH.H₂ O;

0.738 g of Boc-Ser(Bzl)-OH; and

0.586 g of Boc-Asn-OH;

the following polypeptide is obtained:

H-Asn-Ser-Leu-Val-Tyr-Gly-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-OH in goodyield as the acetate salt.

EXAMPLE 9 Maleimide Conjugated Bovine Gamma Globulin and Bovine SerumAlbumin (M-BGG/M-BSA)

A. A 20-mg portion of bovine gamma globulin (BGG) in 5 ml of pH 6.73phosphate buffer is treated with 4 mg of succinimidyl4-(p-maleimidophenyl)butyrate (SMPB, Pierce Chem Co.) in 100 μl of DMF.The mixture is stirred at 25° C. for 1.5 hr and centrifuged. Thesupernatant liquid is desalted on a Sephadex G-25 column by elution withH₂ O the first UV absorbing peak (Abs 2.01 OD at 280 nm) is pooled toyield 20 ml of standard solution of M-BGG[4-(p-maleimidophenyl)butryamido-BGG].

Similarly, by replacing the BGG with BSA is obtained the correspondingM-BSA.

EXAMPLE 10 Human Somatotropin (Growth Hormone) Fragment Conjugates(HGH-M-BGG/HBH-M-BSA)

A 2 mg sample of H-Tyr-Gly-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-Leu-Cys-OH isdissolved in 10 ml of standard solution of M-BGG and stirred at 25° C.overnight. The reaction product (wherein the --SH of the cysteine hasadded to the carbon-carbon double bond) is purified by gel permeationchromatograph on a Sephadex G-25 column by elution with H₂ O. The firstUV absorbing peak is pooled and lyophilized to give the proteinconjugated human somatotropin conjugate (immunogen) as a white powder.Amino acid analysis of the product indicated about 20 molecules of thepolypeptide fragment per molecule of BGG in the conjugate.

Similarly, by replacing the M-BGG by M-BSA is obtained the HGH-M-BSAconjugate.

EXAMPLE 11 Human Growth Fragment Conjugate

By replacing the polypeptide of Example 5 containing the eight aminoacid sequence with a stoichiometrically equivalent amount of thepolypeptide of Example 10 and completing the procedure of Example 10,there is obtained the corresponding polypeptide-M-BGG orpolypeptide-M-BSA conjugate.

EXAMPLE 12 Human Growth Hormone Fragment Conjugate

By replacing the polypeptide of Example 6 containing the fourteen aminoacid sequence with a stoichiometrically equivalent amount of the peptideof Example 10 and using the procedure of Example 10, there is obtainedthe corresponding polypeptide-M-BGG conjugate or polypeptide-M-BSAconjugate.

EXAMPLE 13 Human Growth Hormone Fragment Conjugate

By replacing the polypeptide of Example 7 containing the twenty aminoacid sequence with a stoichiometrically equivalent of the peptide ofExample 10 and using the procedure of Example 10, there is obtained thecorresponding polypeptide-M-BGG conjugate or polypeptide-M-BSAconjugate.

EXAMPLE 14 Human Growth Hormone Fragment Conjugate

By replacing the polypeptide of Example 8 containing the fourteen aminoacid sequence with a stoichiometrically equivalent amount of the peptideof Example 10 and using the procedure of Example 10, there is obtainedthe corresponding polypeptide-M-BGG conjugate or polypeptide-M-BSAconjugate.

EXAMPLE 15 Human Placental Lactogen (Somatomamotropin)-Conjugates(HPL-M-BGG/HPL-M-BSA)

A 2 mg sample of H-Tyr-Asp-Thr-Ser-Asp-Ser-Asp-Asp-Tyr-His-Leu-Cys-OH isdissolved in 10 ml of the standard solution of M-BGG and stirred at 25°C. overnight. The reaction product is purified by gel permeationchromatography on a Sephadex G-25 column by elution with H₂ O. Theearliest eluting UV absorbing (280 mm) peak is pooled and lyophilized toyield the protein carrier coupled to the human placental lactogenfragment. Amino acid analysis of the product indicated that about 20molecules of the polypeptide fragment is coupled to each molecule ofM-BGG.

Similarly, by replacing the M-BGG with M-BSA, there is obtained theHPL-M-BSA conjugate.

EXAMPLE 16 Human Prolactin Fragment Conjugates(HP-N-BGG/HP-N-BSA/HP-C-BGG/HP-C-BSA)

A 7 mg portion of ##STR7## (Human Prolactin C-terminus disulfide loop;HP-C) is treated with 15 mg of BGG and 10 mg ofN-ethyl-N'-dimethylaminopropylcarbodiimide in 5 ml of NaOAc buffer (pH4.65).

The material is desalted by gel permeation chromatography on a SephadexG-25 column in H₂ O. The first ultraviolet absorbing peak is pooled andlyopholized to yield HP-N-BGG or HP-C-BGG as a white powder.

Similarly, by replacing the BGG by BSA one obtains the correspondingHP-N-BSA or HP-C-BSA conjugates.

EXAMPLE 17 Injection of Host with Peptide Immunogen

A portion of the polypeptide conjugate is dissolved in 0.9% saline andemulsified with complete Freunds' Adjuvant (mineral oil containing wholemycobacteria plus emulsifier) by the "two syringe technique" recommendedby the supplier (Gibco). The mixture is injected subcutaneously into thehost animal at multiple sites. The animal is boosted with immunogen inIncomplete Freund's Adjuvant (IFA) at several week intervals until thedesired immunological response is achieved.

For example, a 2 mg sample of the human Ifγ-BGG conjugate is dissolvedin 1 ml of saline and emulsified with 3 ml of Freunds Complete Adjuvant.The resultant vaccine is divided into 4 portions and a 2.2 kg femalerabbit is injected subcutaneously in each shoulder and hip region. Theanimal is boosted at week 3 with 0.1 mg of conjugate in 1/2 ml of IFA bysubcutaneous injection as above. After a further 3 weeks the animal isbled to obtain serum for the ELISA assay and 1 week later is boostedagain. The pattern of boost, wait 3 weeks, bleed, wait 1 week, boost iscontinued through 5 cycles.

The blood sera collected is allowed to clot at 25° C. for several hours.The clear serum is separated from the clot by centrifugation and isdiluted with 0.9% saline to the desired concentration.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin this art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, or composition of matter, process,process step or steps, or the present objective to the spirit and scopeof the invention. In addition, many modifications may be made to adopt aparticular situation, material, or composition of matter, process,process step or steps, or the present objective to the spirit of thisinvention without departing from its essential teachings.

What is claimed is:
 1. The polypeptide which has the amino acidsequence, H-Tyr-Gly-Ala-Ser-Asp-Ser-Asn-Val-Tyr-Asp-Leu-Cys-OH, and thepharmaceutically acceptable salts thereof.
 2. The polypeptide which hasthe amino acid sequence,H-Tyr-Asp-Thr-Ser-Asp-Ser-Asp-Tyr-His-Leu-Cys-OH, and thepharmaceutically acceptable salts thereof.
 3. The polypeptide which hasthe amino acid sequence,H-Leu-Pro-Ile-Cys-Pro-Gly-Gly-Ala-Ala-Arg-Cys-Gly-OH, and thepharmaceutically acceptable salts thereof.
 4. The polypeptide which hasthe amino acid sequence, H-Gly-Cys-Arg-Ile-Ile-His-Asn-Asn-Asn-Cys-OH,and the pharmaceutically acceptable salts thereof.